A new assay to measure the biological activity of a lethal neurotoxin could help scientists develop therapeutic inhibitors of the poison, say US chemists.

If ingested or inhaled Botox can be lethal, causing the muscle paralysis illness botulism

Botulinum neurotoxin A is the active ingredient in Botox, the cosmetic injection used to smooth out wrinkles. But if ingested or inhaled, the toxin's muscle-relaxing effect can be lethal, causing the muscle paralysis illness botulism. This has prompted fears that it could be used as a biological weapon. Current treatments are ineffective against the toxin once it has entered the cells, and only mechanical respirators can keep a patient alive once toxin stops them breathing. Such systems would be overwhelmed in the event of a bioterrorism attack, says Kim Janda and colleagues at the Scripps Research Institute in La Jolla, hence the need for better treatments.

In order to assess the effectiveness of potential inhibitors of the toxin, reliable methods to measure their activity are needed. Current tests are not accurate enough for finer analysis, such as determining the mechanism of inhibition, says Janda. To improve sensitivity and reproducibility, Janda has developed a new assay based on LC-MS - liquid chromatography followed by mass spectrometry.

The new assay uses a peptide mimic of the substrate the neurotoxin targets in the body. The toxin slices the peptide into two pieces. The shorter section can be isolated from the reaction mixture by high performance LC and then quantified against an internal standard using a mass spectrometer. The team used this assay to confirm the potency of an inhibitor they had developed to block the toxin.

'Our aim is to identify a stable, potent and specific inhibitor which could eventually be used in clinical practice,' says Janda. 'The biggest challenge undoubtedly lies in the impossibility to conduct standard clinical trials.'

'This is a useful and robust approach suitable for screening protease inhibitors,' says Linda Lawton, who studies naturally occurring toxins at the Robert Gordon University in Aberdeen, UK. 'It is great to see that they have not only developed the assay but clearly shown its value in their successful characterisation of a potent new inhibitor.'

James Mitchell Crow